In vitro Propagation and Molecular Characterization of Hawthorn species (Crataegus azarolus L.) in Rural Damascus

Jehad Dumireih
General Commission for Scientific Agricultural Research-
Biotechnology Department
Tishreen University, Faculty of Agriculture
 2009

Abstract

In the present study, factors affecting in vitro multiplication of Hawthorn Crataegus azarolus L. have been studied as a contribution in expanding its cultivation mainly in the dry areas as well as to benefit from its multi-medicinal uses.
Shoot cuttings with single nodes and auxiliary buds, excised from adult trees grown in the field under natural conditions in Rankous, Rural Damascus were used as primary explants which were surface-disinfected using HgCl2 or with sodium or calcium hypochlorite followed by three rinses with sterile distilled water before being cultured in test tubes containing MS medium with its macro-nutrients reduced by half. Sodium hypochlorite at 30% for 15 min., gave best results of sterilization with 50% efficiency.
They were then placed onto MS basal medium containing a combination of growth regulators at different concentrations (BA at 5, 10, 15, or 20µM), each with IBA or NAA at
0.5µM, 30 g/l sucrose and solidified with 7 g/l Agar. pH was adjusted to 5.7 before autoclaving on 121c for 20 min. Multiplication rate of 4-folds was achieved every 4 weeks on MS medium supplemented with 5µM BA+ 0.5µM IBA, with 2.69 cm average shoot height. BA was superior with significant differences to Kin at the same concentration which produced only 1.15 new shoots with 1.9 cm average height. Increasing BA concentration to 15 and 20µM resulted in decreasing of shoot height. High BA concentrations also induced callus formation at shoot bases. However, shoots grown on media with Kin had relatively bigger leaf surface but with decreasing shoot number. Average shoot number increased until sixth subculture with multiplication rate of 6 shoots every 4 weeks.
Rooting was achieved within 4 weeks by transferring 2-3 cm shoots onto MS agar-gelled media containing IBA at a conc. of 14.70µM, with a maximum rooting efficiency of 45%.
Rooted plantlets were transplanted into pots with a mixture of 2:1 (v/v) peat: perlite and covered with plastic bags and acclimatized gradually to field conditions throughout 4 weeks. Acclimatized plantlets were transferred to greenhouse for further acclimatization before transplanted in the field with 60 % efficiency.
On the other hand, Randomly Amplified Polymorphic DNA (RAPD) technique was employed in the present study for identifying 27 wild hawthorn genotypes collected from 15 different areas around Damascus countryside and revealing their genetic relationships.
Leaves were collected from wild hawthorn trees for DNA extraction. The DNA isolation procedure was based on a CTAB method with substantial modifications. Polymorphism of investigated genotypes was observed with 52 out of 64 tested RAPD primes which gave repeatable polymorphic product producing a total of 507 bands, 256 of them were polymorphic and 251 monomorphic. The level of polymorphism amounted to 48.37% with an average of 9.75 bands/primer and an average of 4.82 polymorphic bands/primer. Relationship was determined on the base of polymorphic products analysis and presented in form of dendrograms (UPGMA percent method).
There was no correlation between the genotypes which has a close genetic relationship and the regions collected from, where samples of one region were distributed all over the dendrogram.
The current study contributes to preserving biodiversity of hawthorn, where it enables the characterization of genotypes and then propagation of valuable ones to extend their cultivation, especially because some of them are neglected and endangered by extinction.